Supported aldehydic silanes and method of manufacture

ABSTRACT

A method for producing a derivatized aldehydic support matrix material includes activating surface hydroxyl groups on the support matrix material and reacting the activated hydroxyl groups with an aldehydic alkoxy silane. The derivatized aldehydic support matrix material produced is useful for immobilizing bio-molecules in biological applications.

FIELD OF THE INVENTION

[0001] The present invention relates generally to derivatized solidsupports. More particularly, the present invention relates to solidsupports derivatized with silanes containing aldehydic functionalitiesand their use in biological applications.

BACKGROUND OF THE INVENTION

[0002] The use of immobilized bio-molecules is an essential techniquerequired for many biological applications. A common method used toimmobilize bio-molecules is by reaction of the primary amine groups ofthe biological molecules with an aldehyde functionality that is bondedto a solid support matrix.

[0003] A popular method of introducing aldehydes to the solid supportmatrix is through the activation of an amine-functionalized surface witha glutaraldehyde solution. Although popular, this method has severaldisadvantages. First glutaraldehyde is an unstable compound that isdifficult to purify. Additionally, two Schiff bases are present in thecovalent linkage of the bio-molecule to the support. Additionally, theSchiff base linkage of the glutaraldehyde to the support is susceptibleto hydrolysis and thus may lead to ligand leaching. Treatment with areducing agent such as sodium borohydride or sodium cyanoborohydride toremove the Schiff bases can be performed, but this adds an additionalstep to the process.

[0004] There remains a need for a solid support matrix with bondedaldehydic functionalities for immobilizing bio-molecules that is stableand can be produced in a simple process.

[0005] Accordingly, it is desirable to provide a solid support matrixcontaining bonded aldehydic functionalities that is stable and can beused to immobilize bio-molecules. It is further desirable to provide asimple method for producing such a solid support matrix. It is stillfurther desirable to provide an apparatus and method for using a solidsupport matrix with aldehydic functionalities to immobilizebio-molecules for biological applications.

SUMMARY OF THE INVENTION

[0006] The present invention is directed to a derivatized solid supportmatrix containing bonded aldehydic functionalities, that is stable andcan be used to immobilize bio-molecules, as well as to a method forproducing such a derivatized solid support matrix.

[0007] The present invention is further directed to an apparatus andmethod for using a derivatized solid support matrix with aldehydicfunctionalities to immobilize bio-molecules for biological applications.

[0008] In accordance with one embodiment of the present invention, amethod of producing a derivatized matrix material with aldehyde groupsis provided. A raw support matrix material having a surface area withhydroxyl groups occurring on the surface area is activated with an acid.The activated support material is then exposed to an alkoxy aldehydicsilane to produce a derivatized matrix material. The support matrixmaterial can be selected from a number of materials, including but notlimited to glasses, agarose, silica, alumina, glass-coated ELISA plates,resin, nickel, aluminum, zinc and paramagnetic iron. The support matrixmaterial may have naturally occurring hydroxyl groups, or the hydroxylgroups may be introduced artificially. A number of mono, di and trialkoxy aldehydic silanes may be used for producing a derivatized matrixmaterial with aldehyde groups. The alkoxy aldehydic silane is preferablya trialkoxy aldehydic silane.

[0009] In another embodiment of the present inventions, an aldehydicderivatized matrix material comprises a support matrix material having asurface area, and a siloxane coating is disposed on at least a portionof the surface area. The siloxane coating may be a mono-layer or a crosslinked siloxane polymer coating. The siloxane coating has a plurality oforganic substituents containing aldehydic functional groups pendanttherefrom. The organic substituents are bound to the siloxane coatingvia carbon-silicon covalent bonds.

[0010] In accordance with another embodiment of the invention, thealdehydic derivatized support matrix material can be incorporated intoan apparatus for immobilizing bio-molecules for biological applications.Such biological applications include, but are not limited to,combinatorial chemistry, molecular biology, ELISA (Enzyme-LinkedImmunosorbent Assay) plates, and cell sorting and identification.

[0011] There are additional features of the invention that will bedescribed below and which will form the subject matter of the claimsappended hereto.

[0012] It is to be understood that the invention is not limited in itsapplication to the details of construction and to the arrangements ofthe components set forth in the following description or illustrated inthe drawings. The invention is capable of other embodiments and of beingpracticed and carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein, as well as theabstract included below, are for the purpose of description and shouldnot be regarded as limiting.

[0013] As such, those skilled in the art will appreciate that theconception upon which this disclosure is based may readily be utilizedas a basis for the designing of other structures, methods and systemsfor carrying out the several purposes of the present invention. It isimportant, therefore, that the claims be regarded as including suchequivalent constructions insofar as they do not depart from the spiritand scope of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0014] One aspect of the present invention provides a compositioncomprising a derivatized solid support matrix material having a surfaceand a siloxane coating on at least a portion of its surface, linkedthrough Si—O bonds. The siloxane coating comprises a plurality ofsilicon atoms that are either individually mono-linked to the solidsupport matrix or cross-linked to each other and further bonded to thesupport matrix through Si—O bonds to form a siloxane polymer. Each Siunit in the coating has a pendant aldehyde containing organicsubstituent, bound to the siloxane coating via carbon-silicon covalentbonds. The siloxane coating is further bound to the solid support matrixmaterial through Si—O—M bonds, wherein M is the support matrix material.

[0015] According to one embodiment of the composition, the siloxanecoating comprises a multi layer siloxane polymer 1 to 10 Si units deep,preferably 2 to 5 units deep. When viewed in cross section, thisembodiment has the general structure:

[0016] where R is an aldehyde containing organic substituent. R¹ is C₁to C₃₀ alkyl, C₁ to C₃₀ alkenyl, phenyl, naphthyl or hydrogen, and M isthe support matrix material. It should be recognized that in thisembodiment the placement of the individual Si units is random withrespect to the support matrix material. The example given is forillustrative purposes only and is not meant to limit the scope of theinvention.

[0017] In an alternate embodiment, the individual Si units are crosslinked to form a siloxane polymer 1 Si unit deep, which when viewed incross section, has the structure:

[0018] where R is an aldehyde containing organic substituent and M isthe support matrix material.

[0019] In a further embodiment, the individual Si units are mono linkedto the solid support matrix material, which when viewed in cross sectionwould have the structure:

[0020] where R is an aldehyde containing organic substituent. R¹ isalkyl or alkenyl containing 1 to 30 carbon atoms, phenyl, naphthyl orhydrogen and M is the support matrix material.

[0021] The aldehyde containing organic substituent, R, may be a straightchain, branched or cyclic alkane containing from 1 to 30 carbon atoms.Alternatively, R may be aromatic or some other unsaturatedaldehyde-containing hydrocarbon. For example, wherein R is formaldehyde,a single layer polymer coating would have the following structure whenviewed in cross section:

[0022] In an alternative embodiment the aldehydic organic substituentspendant from the Si units may vary between the individual Si units, asshown by:

[0023] where R′, R″ and R′″ may all be different aldehyde containingorganic substituents.

[0024] Suitable materials for the solid support matrix include, but arenot limited to glasses, agarose, silica, alumina, glass-coated ELISAplates, resin, nickel, aluminum, zinc and paramagnetic iron. Preferably,the solid support matrix material comprises silica. The support matrixmaterial may be granular or in the form of beads. The support may alsobe a glass, metal or ceramic slide, or granular or bead materialsupported on a glass, metal or ceramic slide.

[0025] Another aspect of the current invention provides a method forproducing a derivatized matrix support material as described above. Inthis aspect of the invention, a raw solid support matrix material isprovided. The raw solid support matrix material has hydroxyl groupsoccurring on its surface area. The hydroxyl groups may be naturallyoccurring or may be artificially introduced by methods well understoodby those skilled in the art. Such methods include, but are not limitedto treatment with aqueous base, plasma treatments or corona treatments.Preferably, the solid support matrix material is a silica gel.

[0026] The hydroxyl groups on the solid support matrix material may beactivated using an acid. Preferably, a suspension of the matrix materialis formed in an organic solvent and an aqueous acid is added to thesuspension. Preferably the organic solvent is a non-polar solvent, suchas hexanes or n-heptane. The acidified suspension is then mixed andallowed to equilibrate.

[0027] An aldehyde functionality is introduced via an alkoxy aldehydicsilane. The alkoxy aldehydic silane may be a single species or mixtureof species selected from the group having the general formulas:

[0028] where R¹ is an alkyl or alkenyl containing 1 to 30 carbon atoms,a phenyl, a naphthyl, or is a covalent bond. R², R³ and R⁴ areindependently alkyl or alkenyl containing 1 to 30 carbon atoms, phenyl,naphthyl, silyl or hydrogen. In a preferred embodiment, the alkoxysilane is a trialkoxy silane according to structure 3, and none of R²,R³ or R⁴ is hydrogen. In a more preferred embodiment, R², R³ and R⁴ areequivalent. Preferred R², R³ and R⁴ groups are methyl, ethyl and propyl.Preferred R¹ groups are straight chain alkanes having 1 to 10 carbonatoms.

[0029] The alkoxy silane is added to the acidified suspension andallowed to react with the activated hydroxyl groups on the surface ofthe matrix material. Preferably, the alkoxy silane is added in smallportions over a period of hours. Most preferably, there is anequilibration period between additions of the alkoxy silane. Forexample, 10 mL of aldehydic alkoxy silane may be added to a suspensionof 30 grams of silica in 0.5 mL portions at intervals of 8 to 12 minutesover a period of 3 to 4 hours. Under the acidic conditions in thesuspension, the alkoxy silane will be hydrolyzed, producing an alcoholand silanol for each alkoxy substituent, following the general formula:

RSi(OR′)₃+3H₂O→RSi(OH)₃+3R′OH

[0030] where R is the aldehyde containing organic substituent. Thesilanol thus produced can then react with the activated hydroxyl groupson the surface of the matrix material to produce the derivatizedmaterial. It will be recognized that the stoichiometry of the reactionwill vary depending on alkoxy aldehydic silane employed.

[0031] Following addition of the alkoxy silane, the now derivatizedmatrix material is preferably collected and washed to remove excessacid, unbound silane and polymers.

[0032] It is recognized that a number of solid support matrix materialscan be used alone, or in combination to achieve a derivatized materialhaving desired qualities. It is also recognized that mixtures of alkoxysilanes having different aldehydic substituents can be used to produce aderivatized material having a wide variety of desired qualities.

[0033] The derivatized aldehydic material thus produced can be used aspart of an apparatus for immobilizing bio-molecules for biologicalapplications. In one embodiment of this aspect of the invention, apolypropylene column is used to contain an aldehydic derivatized supportmatrix material according to the current invention. The column is openat one end and has a porous bed support at the opposite end forsupporting the matrix material, while allowing fluid to pass. An exampleof a column suitable for use in this embodiment is the POLY-PREP®conical polypropylene column, available from Bio-Rad Laboratories Inc.

[0034] In another embodiment of this aspect of the invention a columnmay be used that is compatible for use with a centrifuge. In thisembodiment, separation of sample components is affected throughcentrifugal force. An example of a column suitable for use in thisembodiment is the MICRO BIO-SPIN® chromatography columns, also availablefrom Bio-Rad Laboratories Inc. However, it will be recognized that theinvention is not limited to a particular brand of column or a particulardesign, shape or material of construction.

[0035] The apparatus as described in the embodiments above can be usedto immobilize a wide variety of bio-molecules. The general procedurefollowed to immobilize a bio-molecule involves first washing thederivatized support matrix material contained in the column with abuffer solution of appropriate pH. The pH of the buffer solution willvary depending on the bio-molecule to be immobilized and the derivatizedsupport matrix material being used. However, the pH of the buffersolution is preferably in the range of about 4 to about 10, morepreferably from about 8 to about 10. A solution containing thebio-molecule to be immobilized is then added to the column in a buffersolution. The apparatus containing the bio-molecule solution is thenincubated at an appropriate temperature, for a time sufficient toimmobilize at least a portion of the bio-molecule contained in thesolution. Again incubation temperatures and times will vary depending onthe bio-molecule being immobilized and the derivatized support matrixmaterial being used. Following the incubation period, the column isdrained of solution and preferably washed at least once with a freshbuffer solution. Preferably, the buffer solution used is identical tothe solution used to initially wash the column. Washing with buffersolution removes any unbound material from the column. The apparatuscontaining the immobilized bio-molecule may then be further derivatizedor used in an assay as desired.

[0036] In another embodiment of this aspect of the invention, thesupport material is a glass coated ELISA (Enzyme-Linked ImmunosorbentAssay) plate. In this embodiment, the test bio-molecule is attachedcovalently to the ELISA plate. A rapid test can then be performed, wherean antibody or antigen is coupled to an enzyme as a means for detectingan antigenic match.

[0037] Other applications where the current invention may be employedinclude combinatorial chemistry; tethered amine modifiedoligonucleotides for PCR (Polymerase Chain Reaction); RNAisolation/purification; DNA micro arrays, probes and genome chips; cellsorting and identification.

[0038] The following examples demonstrate the method of producing aderivatized support material according to the current invention and amethod of using the invention for immobilizing bio-molecules.

EXAMPLE 1

[0039] In a 250 mL beaker, 25 grams of raw 40-60 μm silica gel wassuspended in 250 mL of hexanes. A solution of 0.30 mL of glacial aceticacid in 2.0 mL of water was then added to the suspension. The mixturewas allowed to equilibrate by mixing for 30 minutes. Over a 2-3 hourperiod, 7.5 mL of triethoxy aldehydic silane was added to the suspensionin 0.5 mL aliquots. After each addition, the suspension was purged withnitrogen and recovered. Following the additions, the suspension wasallowed to mix for five hours. The silica was collected and washed with600 mL of isopropyl alchohol and one 1 L of deionized water. Theresulting aldehydic silica was stored in a glass container and immersedin N₂ purged deionized water under nitrogen at 4° C. This procedure wasrepeated three times.

EXAMPLE 2

[0040] The four derivatized aldehydic silicas produced in Example 1 wereeach analyzed in duplicate to quantify of polymeric coating on thesilica. The organic loading of the silicas was determined using thefollowing method. Samples of each of the four silicas produced inExample 1 were taken and dried under nitrogen at 103° C. for four hoursand then cooled in a desiccator. The dried material was weighed and thenignited in a furnace at 950° C. Igniting the samples caused all of theorganic material to be volatilized, leaving only SiO₂. The organicloading of the samples can be expressed as:

% organic loading=[1−(wt. ash/wt. sample)]×100

[0041] where “wt. sample” is the mass of the sample before ignition and“wt. ash” is the mass of the sample after ignition. A silica gel blankwas also ignited under the same conditions as a control. The results areshown in TABLE 1. TABLE 1 Organic Loading Sample Average of 2 trials 18.20% 2 8.13% 3 8.23% 4 8.29% average 8.21% silica blank  3.5%

[0042] The overall average organic loading found on the derivatizedsilica produced in Example 1 is 8.21%, with a standard deviation of0.067% and a coefficient of variation of 0.82%. In comparison, thesilica gel blank showed a carbon loading of only 3.5%. These valuesdemonstrate the successful attachment of the alkoxy aldehydic silane tothe silica particles.

EXAMPLE 3

[0043] A bio-molecule, Protein A, available from Prozyme, Inc., wasattached to the derivatized support material produced in Example 1 todemonstrate the activity of the silica surface as well as the loadingcapacity of the Protein A. The bio-molecule Protein A is available froma number of natural and artificial sources.

[0044] The Protein A used in this example was produced from the enzymestaphylococcus aureus. A 0.4 mL sample of the aldehydic silica was addedto a POLY-PREP® polypropylene fritted column, available from Bio-Rad.The material was then washed with 5 columns full of 0.0 1M PhosphateBuffer Saline (PBS buffer), pH 7.4, available from Sigma Chemical. Thealdehydic silica was then incubated with 3.41 mg of protein A in PBSbuffer overnight.

[0045] The column was then allowed to drain into a test tube, the volumenoted, and the absorbance of the supernatant at 280 nm and 320 nm wasnoted. The column was then washed with 1 mL of PBS buffer. The wash wascollected and the volume recorded, as well as, the absorbances at 280 nmand 320 nm. The wash procedure was repeated with fresh PBS buffer untilthere was no significant absorbances recorded at 280 nm or 320 nm,insuring that no unbound protein A was present. The amount of protein Apresent in the washes and supernatant was determined as follows:

[0046] mg in solution=((A₂₈₀−A₃₂₀)/0.14)×volume solution

[0047] A₂₈₀=Absorbance at 280 nm

[0048] A₃₂₀=Absorbance at 320 nm

[0049] 0.14 =Extinction coefficient (E^(0.1%)) of protein A

[0050] The absorbance at 320 nm represents light scattering due to thepresence of particles, notably silica, in the solution. These particleswill also cause absorbance at 280 nm. Protein A has a strong absorbanceat 280 nm, but little to no absorbance at 320 nm. By subtracting theabsorbance at 320 nm from the absorbance at 280 nm, a true value of theabsorbance due to protein A is obtained.

[0051] The amount of protein A bound to the silica was calculated bysubtracting the amount of protein A collected in the supernatant andwashes from the amount of protein A originally loaded on the columns(3.41 mg). Four samples of the derivatized silica produced in Example 1were used and yielded the results shown in TABLE 2. TABLE 2 SampleProtein A loading 1 1.59 mg 2 1.50 mg 3 1.51 mg 4 1.56 mg average 1.54mg

[0052] The standard deviation of the four trials was 0.04 mg and thecoefficient of variation was 2.60%. These values demonstrate thesuccessful attachment of protein A to the derivatized aldehydic silica.

EXAMPLE 4

[0053] The activity of the protein A columns was confirmed byqualitative enzymatic assay. A sample of 0.2 mg of peroxidase conjugatedrabbit IgG whole molecule, available from Rockland, was diluted in 2 mLof cool 0.01M phosphate, 0.5M NaCl PBS coupling buffer. The enzymesolution was added to the protein A columns. A negative controlconsisted of the enzyme solution added to 0.4 mL of the derivatizedsilica without protein A attached. The protein A columns and the controlwere then incubated for 1.5 hours at 4° C. with gentle end-to-endmixing.

[0054] After incubation, the columns were drained and washed with PBSbuffer. The washes were collected and the absorbances at 320 nm and 280nm read. The columns were washed until there was no significantabsorbance at either 280 nm or 320 nm, ensuring that no unboundconjugated IgG remained.

[0055] Next, 2.90 mL of 9.1 mM 2,2′-Azino-bis(3-Ethylbenzthiazoline-6-sulfonic Acid) (ABTS) in 100 mM potassiumphosphate buffer, pH 5.0, and 0.05 mL of 0.3% (w/w) hydrogen peroxidesolution in deionized water, were added to each of the columns.Peroxidase will catalyze the following reaction:

H₂O₂+ABTS→H₂O+oxidized ABTS

[0056] Oxidation of ABTS causes the development of a dark blue color,caused by an increase in the absorbance at 405 nm. During thequalitative assay, the protein A columns displayed instant and intensecolor development, while the negative control showed only an extremelyslight color change over several minutes. The intense blue color in theprotein A columns indicates the presence of bound active peroxidaseconjugated IgG. The slight color change in the control is most likelydue to minute amounts of peroxidase conjugated IgG that wasnonspecifically bound to the aldehydic silica and not removed by therepeated washes.

EXAMPLE 5

[0057] Example 1 was repeated using 4% cross linked agarose in place ofsilica. Two duplicate samples of agarose were derivatized following thesame general methodology as in Example 1, with the followingmodifications; 1) samples of 4% cross linked agarose were used in placeof silica, 2) the samples were suspended in 50 mL of hexanes, 3) 20 mLof hexanes were added 3 hours into the reaction, 4) after the additionof the siloxane, the reaction was allowed to run 2 hours.

[0058] Organic loading testing could not be performed with agarose.Binding of protein A to the derivatized agarose was performed on columnscontaining 0.5 mL of derivatized agarose. Protein A loading values of1.53 and 1.34 mg of protein A per 0.5 mL of derivatized agarose wereobtained.

[0059] The forgoing examples demonstrate the successful binding ofaldehydic alkoxy silanes to solid support matrix materials to produce aderivatized aldehydic matrix material. Also demonstrated is thesuccessful binding of bio-molecules to the derivatized material.

[0060] It will be apparent to those skilled in the art that theinvention of the present application is applicable to uses other thanthose demonstrated above. It is recognized that a wide variety of solidsupport materials having surface hydroxyl groups, or capable of havinghydroxyl groups introduced, can be used. Further, it is recognized thata wide variety of aldehydic alkoxy silanes can be used, and that thederivatized materials produced therefrom could be used with a widevariety of bio-molecules in a number of biological applications. Hence,all of these equivalents are considered within the scope of the currentinvention.

What is claimed is:
 1. A method of producing a derivatized matrixmaterial with aldehyde groups, said method comprising, providing a rawmatrix material, said raw matrix material having a surface area andhydroxyl groups occurring on said surface area; activating said hydroxylgroups with an acid; exposing said raw matrix material to an alkoxyaldehydic silane to produce a derivatized matrix material, wherein saidalkoxy aldehydic silane has a structure selected from the group:

wherein, R¹ is alkyl from C₁ to C₃₀, phenyl, naphthyl or a covalentbond, and R², R³ and R⁴ are independently alkyl or alkenyl from C₁ toC₃₀, phenyl, naphthyl or silyl,

wherein, R¹ is alkyl from C₁ to C₃₀, phenyl, naphthyl or a covalentbond, and R², R³ and R⁴ are independently alkyl or alkenyl from C₁ toC₃₀, phenyl, naphthyl or silyl, and

wherein, R¹ is alkyl from C₁ to C₃₀, phenyl, naphthyl or a covalentbond, and R², R³ and R⁴ are independently alkyl or alkenyl from C₁ toC₃₀, phenyl, naphthyl or silyl.
 2. A method according to claim 1,wherein said alkoxy aldehydic silane is a trialkoxy aldehydic silanehaving the structure:

wherein, R¹ is C₁ to C₃₀ alkyl, C₁ to C₃₀ alkenyl, phenyl, naphthyl or acovalent bond, and R², R³ and R⁴ are C₁ to C₃₀ alkyl, Cl to C₃₀ alkenyl,phenyl or naphthyl, and R², R³ and R⁴ are the same.
 3. A methodaccording to claim 1, further comprising forming a suspension by addingan organic solvent to said raw matrix material, and activating saidhydroxyl groups by adding an aqueous acid to said suspension, andrecovering and equilibrating said suspension for a period of at leastabout 15 minutes; adding said alkoxy aldehydic silane to said suspensionin small aliquots over a period of at least about 1 hour; andrecovering, and then washing said derivatized matrix material with asolvent.
 4. A method according to claim 3, wherein said alkoxy aldehydicsilane is a trialkoxy aldehydic silane having the structure:

wherein, R¹ is C₁ to C₃₀ alkyl, C₁ to C₃₀ alkenyl, phenyl, naphthyl or acovalent bond, and R², R³ and R⁴ are C₁ to C₃₀ alkyl, C₁ to C₃₀ alkenyl,phenyl or naphthyl, and R², R³ and R⁴ are the same.
 5. A methodaccording to claim 1, wherein said matrix material is selected from thegroup consisting of glasses, agarose, silica, alumina, glass-coatedELISA plates, resin, nickel, aluminum, zinc and paramagnetic iron.
 6. Analdehydic derivatized matrix material comprising, a support matrixmaterial having a surface area, and a siloxane coating disposed on atleast a portion of said surface area, and said siloxane coating having aplurality of organic substituents containing aldehydic functional groupspendant therefrom.
 7. An aldehydic derivatized matrix material accordingto claim 6, wherein said support matrix material is selected from thegroup consisting of glasses, agarose, silica, alumina, glass-coatedELISA plates, resin, nickel, aluminum, zinc and paramagnetic iron.
 8. Analdehydic derivatized matrix material according to claim 6, wherein saidaldehydic functional groups are bonded to said siloxane coating bycarbon-silicon bonds.
 9. An aldehydic derivatized matrix materialaccording to claim 8, wherein said siloxane coating is a cross linkedsiloxane polymer.
 10. An aldehydic derivatized matrix material accordingto claim 9, wherein said cross linked siloxane polymer has the generalformula:

where M is the support matrix material, n is an integer from 1 to about10,000 and R⁵ is an organic substituent containing an aldehydicfunctionality.
 11. An aldehydic derivatized matrix material according toclaim 8, wherein said siloxane coating is a mono layer having theformula:

where M is the support matrix material, n is an integer from 1 to about10,000 and R⁵ is an organic substituent containing an aldehydicfunctionality and R⁶ and R⁷ independently alkyl or alkenyl from C₁ toC₃₀, phenyl or naphthyl.
 12. A method for immobilizing bio-molecules,said method comprising: a) providing a column containing aldehydicderivatized matrix material comprising a support matrix material havinga surface area, and a siloxane coating disposed on at least a portion ofsaid surface area; said siloxane coating having a plurality of organicsubstituents containing aldehydic functional groups pendant therefrom;b) washing said column with a buffer; c) adding to said column asolution containing bio-molecules to be immobilized, and d) incubatingsaid column to immobilize at least a portion of said bio-molecules. 13.A method according to claim 12, wherein said support matrix material isselected from the group consisting of glasses, agarose, silica, alumina,glass-coated ELISA plates, resin, nickel, aluminum, zinc andparamagnetic iron.
 14. A method according to claim 12, wherein saidaldehydic functional groups are bonded to said siloxane coating bycarbon-silicon bonds.
 15. A method according to claim 14, wherein saidsiloxane coating is a cross linked siloxane polymer.
 16. A methodaccording to claim 15, wherein said siloxane polymer has the generalformula:

where M is the support matrix material, n is an integer from 1 to about10,000 and R⁵ is an organic substituent containing an aldehydicfunctionality.
 17. A method according to claim 14, wherein the siloxanecoating is a mono layer having the general formula:

where M is the support matrix material, n is an integer from 1 to about10,000, R⁵ is an organic substituent containing an aldehydicfunctionality and R⁶ and R⁷ independently alkyl or alkenyl from C₁ toC₃₀, phenyl or naphthyl.
 18. An apparatus for immobilizingbio-molecules, said apparatus comprising, a hollow column, said columnbeing adapted for receiving a matrix material to be filled into saidcolumn; an aldehydic derivatized matrix material disposed in saidcolumn, said derivatized matrix material comprising, a support matrixmaterial having a surface area, and a siloxane coating disposed on atleast a portion of said surface area; and said siloxane coating having aplurality of organic substituents containing aldehydic functional groupspendant therefrom.
 19. An apparatus according to claim 18, wherein saidhollow column has a top end and a bottom end, said top end being openfor receiving said matrix material, and said bottom end having a frittedplug, said fritted plug having a porosity such that said fritted plugpermits the exit of fluids from said hollow column.
 20. An apparatusaccording to claim 18, wherein said hollow column is a microtube, saidmicrotube having a top end and a bottom end, said top end having anopening and a cap, and said microtube being generally adapted for usewith a centrifuge.
 21. An apparatus according to claim 18, wherein thehollow column is adapted to accommodate a raw solid support matrixmaterial, an aqueous acid and an alkoxy aldehydic silane.
 22. Anapparatus according to claim 18, wherein said support matrix material isselected from the group consisting of glasses, agarose, silica, aluminaglass-coated ELISA plates, resin, nickel, aluminum, zinc andparamagnetic iron.
 23. An apparatus according to claim 18, wherein saidaldehydic functional groups are bonded to said siloxane coating bycarbon-silicon bonds.
 24. An apparatus according to claim 23, whereinsaid siloxane coating is a cross-linked siloxane polymer.
 25. Anapparatus according to claim 24, wherein said siloxane polymer has theformula:

where M is the support matrix material, n is an integer from 1 to about10,000 and R⁵ is an organic substituent containing an aldehydicfunctionality.
 26. An apparatus according to claim 23, wherein saidsiloxane coating is a mono layer having the formula:

where M is the support matrix material, n is an integer from 1 to about10,000, R⁵ is an organic substituent containing an aldehydicfunctionality and R⁶ and R⁷ independently alkyl or alkenyl from C₁ toC₃₀, phenyl or naphthyl.